EPIGENETIC AGE CLOCK TEST REPORT

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Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20 Test: TruAge™ | Sample ID:
BL305
Collection Date: 2/21/20
Report Date: 4/12/20

Hi Jane,

Thanks so much for taking the TruAge™ test by TruDiagnostic™. TruDiagnostic™ is a company
that has been built on one premise. We want to be able to read your DNA methylation
patterns so that we can help you live longer, better quality life. In the report below, we will
explain everything about our test including why it is important and how you can use this
metric to live a healthier life.

By purchasing TruAge™, you have now unlocked a lifetime of information about yourself.
As we get better at reading each methylation spot on your DNA, and the outcomes that
each spot is correlated to, we will continue to update you on the information and what
it tells us about you. You are one of the first to have your DNA read and interpreted by
our innovative algorithms. We are thankful to you for adding to the growing science and
innovation around these areas.

Hopefully this will be the first of many times we report our hard work to you and help you
unlock a longer, healthier life.

Thanks,
The TruDiagnostic™ Team

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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

TABLE OF CONTENTS

4 What is Epigenetics? 14 Is Aging A Disease?

5 What is Biological Age? 15 How Do I Slow My Aging?

6 Why Methylation Markers? 16 The TruAge™ Treatment Framework

7 How Was This Test Created? 64 My TruAge™ Framework

8 Your TruAge™ Summary 67 Current Questions

9 Your Biological vs Chronological Age 68 Future Test

10 Terms You Should Know 69 Key Terms

11 Your EpiAge™ Ratio 71 References

12 How Do You Compare? 78 Contact

13 Your TruAge™ Over Time

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

WHAT IS EPIGENETICS
and how is it different than genetics?

Epi - is a greek prefix for “above”. Genetics is the

study of our DNA. Together, epigenetics means the
study of things above and beyond the genome.
Genetic Testing VS Epigenetic Testing
This means we are studying the changes to your
DNA and how it actually affects the body instead 23andMe
and Similar Testing
of what the DNA could possibly do or mean.

It is often more useful than genetics because it allows Measures the

Genetic Code
us to see how the genetic material in your body
behaves instead of just seeing what it contains.
Traditional genetics is like looking at a light bulb and its
Reports Health
components but not knowing if the bulb is producing Risks
light. Epigenetics lets us know if the light bulb is on
or off.
Measurement of How
Genes Are Expressed
The link between epigenetics and health has been
linked through biological age. This is important because
aging is THE leading risk factor for multiple chronic
Able to Influence with
diseases and disorders. Therefore, finding a way to Lifestyle Changes
slow the biological aging process is essential. Through
epigenetics, TruAge™ does just that. Our epigenetic
clock is the most accurate measurement of biological Unique Algorithms
For Health Insights
age and age-related disease risk!

Epigenetic aging can be reversed, so it is crucial to

Measures a Value
understand DNA methylation changes through that You Can Improve
utilizing TruAge™. Since we know that it can be Over Time

reversed, we can apply changes to our lifestyles and

use TruAge™ to show that we are reducing YOUR risk
of incidence of disease and death.

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

WHAT IS BIOLOGICAL AGE

and why is this important to know? CHRONOLOGICAL AGE
The number of years that have
passed since birth. This cannot
Everyone knows their chronological age. Chronological be influenced by lifestyle and
age is the number of candles that are on top of your eating habits
cake and the birthdays that you celebrate (or sometimes
don’t!). However, developments in science have
created another measurement of age called biologic age.
This measurement of age is based on years of statistical
research which can predict how healthy you are and
even when you might pass away.

The novel DNA biomarker uses markers on your DNA

called methylation to predict your age. Your biological
age is more accurate at predicting health span (how
healthy you are) and lifespan (how long you will live) than
any previous molecular biomarker, and can be correlated
to aging-related conditions such as Alzheimer’s disease
and cancer. Ideally, everyone would want their biological
age to be less than their chronological age. This means
that you are living a lifestyle that is healthy and will help
you stay free of sickness and disease longer.
BIOLOGICAL AGE
It is a single metric that takes all the important health
How old our cells really are, therefore,
data (weight, sex, medication, exercise frequency, etc.)
our real age. This can be reversed by
about an individual and is able to report back in one
attending to your health.
single metric how healthy you are!

WHY IS THIS IMPORTANT?

When we are born, all of our cells have the same DNA. The cells in your eyes have the same DNA as the cells in your
hair. So what makes our cells different?

The answer is each cell chooses to turn on some genes and turn others off. Your eyes express different genes
than your hair. This is called expression, and it is controlled by the markers we measure with our TruAge™ test.
Unfortunately, as we age, expression can become much harder to regulate. The genes which should be
expressed in your eyes aren’t regulated as well and you start to lose function. In fact, aging is defined as the
progressive loss of function as you age! So what does this have to do with biological age measured by TruAge™?

TruAge™ can report how old your cells and DNA look. Meaning you can measure how likely you are to develop
disease or how long you might live!

Often, knowing how to be the healthiest you can be is difficult. Doctors measure blood levels like cholesterol,
inflammation, and blood sugar. They perform tests such as colonoscopies, vision tests, and physical function tests.
Now, with this single measurement, you can link your health and longevity to a single, simple test which can help you
and your doctor know the best way to address your health concerns in a personalized way!
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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

A recent review six types of potential biological age

estimators:

- Epigenetic Clocks
- Telomere Length
- Transcriptomic-Based
- Proteomic-Based
Why are - Metabolomic-Based
METHYLATION - Composite Biomarkers

MARKERS a The study concluded that the epigenetic clock is the

most promising molecular estimator of biological age.1
better measurement
of Biological Age
Similarly, a comparative review of different forensic
than other factors? methods for age estimation concluded that DNA
methylation is the most promising age-predictive
biomarker.2

Source:
1. Jylhävä, Juulia, et al. “Biological Age Predictors.” EBioMedicine,
vol. 21, 2017, pp. 29–36., doi:10.1016/j.ebiom.2017.03.046.

2. Lee, Hwan Young, et al. “Forensic DNA Methylation Profiling from

Evidence Material for Investigative Leads.” BMB Reports, vol. 49,
no. 7, 2016, pp. 359–369.

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

HOW WAS THIS TEST

CREATED and is it accurate? 850,000 PLACES ON THE DNA

WE LOOK AT OVER 850,000 PLACES ON THE DNA.

HOW DO WE MAKE SENSE OF WHAT WE FIND?
We have the most robust testing available in the world
for biological age. The data we get from your DNA is
40,000 times larger than many competitors. But how
do we know how this data applies to your health?

The answer is we use confusing mathematical models

built by computer learning and artificial intelligence.
This model helps to design a powerful algorithm. It
does this by looking at all the data points we feed it.
Thus far, we have given it almost a million data points
from over 5,000 patients. We also feed it other variables
such as blood tests, imaging data (such as MRI),
genomic data, proteomic data, transcriptomic data
and even other bits of health history.

By looking at all of this data, it is able to find

correlations with incredibly high accuracy and link these
variables to health outcomes. If 2000 patients show
methylation on their DNA at the same place, and 1999
VS
of those patients develop Alzheimer’s, we can say with
a high degree of certainty that that location on the
DNA can help predict risk of Alzheimer’s.
2,500 POINTS COMPETITORS LOOK AT
These mathematical calculations have been performed
with biological aging. By comparing the biological
age and chronological age of a person we can predict
their risk of many many different diseases and even
when they might die themselves!

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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

YOUR TRUAGE™
Summary

Your biological age vs chronological age Your EpiAge™ Ratio

Your TruAge™ Compared to the General Population

Read further to see explanations for each of your results!

Also, see your TruAge™ Treatment Framework on pg 63.

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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

YOUR TRUAGE™
Biological Age vs Chronological Age

TRUAGE™

31.0
7 YEARS
Age 0 Age 96
Our Oldest Patient

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Your biological age is higher than your chronological age.

This is the first of hopefully many tests to measure the

status of your DNA. You are more than your DNA. While tests
like 23andMe might predict risk of certain diseases, TruAge™
can see how much your DNA can be changed through proper
lifestyle changes.

If your TruAge™ is much higher than your chronological age,

don’t worry! There are plenty of things you can do to slow your
aging. If your TruAge™ is under your chronological age, don’t
stop doing what you are doing, but maybe add things which
could make it even better!)

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

TERMS YOU SHOULD KNOW

ADVANCED EPIGENETIC AGING

Advanced Epigenetic aging is having an EpiAge™ ratio
above one. This means through the lens of your DNA
methylation, you are aging quicker than you should!
This is ultimately what we want to avoid as advanced
epigenetic age is correlated to aging diseases and
more negative health outcomes.

IEAA (INTRINSIC EPIGENETIC AGE) AND THE LINK

TO IMMUNOSENESCENCE
As we age, we have other changes than what happens
epigenetically on our DNA. One of the biggest changes
to our health and body is called immunosenescence.
Immunosenescence is when our immune system
becomes weaker and less functional as we age. This
is often seen in the blood by having a fewer number
of naive T cells and a higher number of senescent
T Cells. There are other changes in the number and
percentage of cells that make up the blood as well EEAA (EXTRINSIC EPIGENETIC AGE)
but because of these changes that we see health Extrinsic Epigenetic Age is also important. This is
consequences like older individuals being more likely when we factor immune cells back into the equation.
to die from the flu or COVID-19! [53] Scientifically put, EAA tracks both age-related
changes in blood cell composition and intrinsic
Because the DNA of these cells are found in unequal epigenetic changes. [11]
proportions as we age, we often want to control for this.
In the rest of this report you will see these two
Therefore, IEAA or Intrinsic epigenetic age is when we terms mentioned as they are both useful makers
factor the change of these cell types out of the equation. for health. Although intrinsic measures seem to
In scientific terms, the measure of intrinsic epigenetic exhibit greater consistency across cell types and
age acceleration (IEAA) measures “pure” epigenetic organs, extrinsic measures seem to be better
aging effects that are not confounded by differences suited for assessing age-related decline of tissue
in blood cell counts. [Okazaki et al. 2019] performance as they exhibit stronger predictive
associations with time to death than intrinsic
measures of age acceleration.

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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

YOUR EPIAGE™ RATIO

Biological 31
= EpiAge™
Chronological = 1.29
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You show accelerated aging

Your EpiAge™ ratio is faster than your chronological age.

individuals whose epigenetic age was greater than their
chronological age (i.e., individuals exhibiting epigenetic “age
acceleration”) were at an increased risk for death from all
causes, even after accounting for known risk factors.

Your EpiAge™ ratio is 1.29. This means you age 129% for ev-
ery year. If you lived 20 more years at this rate your age would
be X when you are X.

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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

HOW DO YOU COMPARE

to the general population?

Your TruAge™ Compared to the General Population

This graph shows you where most people would fall on the graph
when comparing their chronological age versus their TruAge™.

One thing to remember is that a majority of our patient population

are receiving this test in a preventative, integrative, functional
medical community. As a result, our population metrics might
be slightly different than those of the general population. That is
because often, the individuals who are being tested can afford the
test and are most likely interested in aging in a healthy manner.
In order to avoid this bias, TruDiagnostic™ actively recruits
participants outside of this population to make sure we have a
good snapshot of all variables such as socioeconomic status,
race, gender, nationality and many others. If you have a
connection to a under represented group who would like to be
involved in this research please let us know!

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

YOUR TRUAGE™ OVER TIME

How to measure your progress

Your 1st TruAge™ & EpiAge™ Ratio Results: Your 2nd TruAge™ & EpiAge™ Ratio Results:

7 YEARS 4 YEARS

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Your EpiAge™ ratio is 1.29. This means you age 129% for every year.
Your EpiAge™ ratio is 1.15 This means you age 112% for every year.
If you lived 20 more years at this rate your age would be 51 when
If you lived 20 more years at this rate your age would be 50 when
you are 44.
you are 46.

Your 3rd TruAge™ & EpiAge™ Ratio Results: Your TruAge™ Ratio Over Time

-2 YEARS

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Your EpiAge™ ratio is .94. This means you age 94% for every year.
If you lived 20 more years at this rate your age would be 48.8
when you are 50.

Key:

Blue Dot: Chronological Age

Yellow Dot: TruAge™

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

IS AGING A DISEASE?

Most scientists and medical professionals describe aging as “a persistent decline in the age-specific fitness
components of an organism due to internal physiological degeneration.” -[24] The longstanding question if old
age is itself a disease has been addressed since ancient times, starting from the Roman playwright Terentius,
who claimed “senectus ipsa est morbus” (old age itself is a disease), and Cicero who some decades later
argued in De Senectute: “pugnandum, tamquam contra morbum sic contra senectutem” (we have to fight
against aging, as we do against a disease). These quotations elegantly summarize a long-held view of aging
and old age!

Aging is the predominant risk factor for most diseases and conditions that limit healthspan. Accordingly,
interventions in animal models that end up in an extension of lifespan often prevent or delay many chronic
diseases. Why? For many years the explanation was that aging per se is a physiological condition, which
favors the onset of many diseases. However, their relationship is likely much more complex. Usually scientists
say this relationship is related via 8 (or 9) hallmarks of aging. [43]

Hallmarks of Aging: TruDiagnostic™ is actively investigating how all of these hallmarks might affect the TruAge™
Epigenetic aging rate.

Due to the fact that aging is the

predominant risk factor for most GENOMIC INSTABILITY
diseases and conditions the limit
lifespan, using this as a target of TELOMERE ATTRITION
optimal health makes sense! PRIMARY HALLMARKS
CAUSES OF DAMAGE
EPIGENETIC ALTERATIONS
Additionally, the specificity and
accuracy of epigenetic tests make LOSS OF PROTEOSTASIS
this a reliable metric to judge health
by limiting aging and therefore DEREGULATED NUTRIENT SENSING

reducing the major risk factors for

disease. Even the world health MITOCHONDRIAL DYSFUNCTION
ANTAGONISTIC HALLMARKS
RESPONSES TO DAMAGE
organization recently added a code to
identify diseases linked with aging! CELLULAR SENESCENCE

We don’t believe that aging is STEM CELL EXHAUSTION

required, and that you can affect the INTEGRATIVE HALLMARKS
CULPRITS OF THE PHENOTYPE
aging process in your own body. We ALTERED INTERCELLULAR
want to help you with that process. COMMUNICATION

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

HOW DO I SLOW MY AGING?

WHAT DOES THE DATA SAY?

Unfortunately, the data around slowing epigenetic
age is very, very new. While this means you are on
the cutting edge of medical therapy, it also means
that interventions to help reverse your epigenetic
age are still being investigated.

With that being said, there have been hundreds of

published trials showing the links between behaviors,
environment, and genetics which you can use as a
guidance to reduce your epigenetic age and therefore
your risk of morbidity and mortality.

We will discuss these in YOUR TruAge™ treatment

framework below!

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

“60% of the determination of

THE TRUAGE™ the aging rate is due to factors
Treatment Framework that you can control!”

We have created the TruAge™ framework to let you see Inner Circle - Intrinsic Aging - The Thread
the biggest areas that affect your EpiAge™ ratio! Much of Life - Genetics, Predispositions
like your epigenetics, this framework is constantly
Unfortunately, there are some aspects of your
updating and changing. We will continue to update
epigenetic aging that aren’t within your control.
your treatment framework with new results that
Usually these things have to do with your underlying
comeout and try and give you suggestions on how to
genetic predispositions or the epigenetic traits
age better. Together, we will use this framework to
passed on by your parents and even grandparents.
tell you what the literature says about the best way
While often times there is little to do about these
to age slowly.

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THE TRUAGE™
Treatment Framework

The Impact To You - Where you Stand

Throughout the rest of this report, you will see the graphic above! This will be a quick signal to let you know how
your demographic information trends according to the data.

The Red Side: Factors Increasing The Green Side: Factors Decreasing
Your Epigenetic Age Your Epigenetic Age

This means that the answers you provided This means that the answers you provided
us, and the results we extrapolated from us, and the results we extrapolated from your
your epigenetic test are associated with epigenetic test are associated with lower
higher epigenetic aging. Oftentimes, this is epigenetic aging. Oftentimes, this is associated
associated with negative health outcomes. with positive health outcomes.

In the paragraphs of text, we will review the This is good news! We still encourage you to
impact and the evidence which has been read the information next to these images.
described in the literature so that you can Often, there is a middle ground or “goldilocks
judge the effect of this trend in yourself and zone” with these metrics. This means that
how you might be able to address this in some behaviors might be good generally,
your TruAge™ Treatment Framework. but bad when done too often. By reading the
explanations, or talking to your physician, you
can see exactly what activities, history, or
actions are beneficial to make efforts to
continue this trend!

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THE TRUAGE™
Treatment Framework

Genetics
Genetics regulate many of the epigenetic modifications
the body has. For instance, the epigenomes of identical
twins are known to be more similar than those of
fraternal twins [78]. This shows that your own genetic
make-up is partly responsible for the way that your
body makes methylation changes to itself. Since you
can’t affect or change it, it is considered an intrinsic
form of epigenetic aging.

Epigenetic Inheritable Changes “The Middle Ground of Nature vs Nurture”

Parental experiences:
Everyone knows that your DNA is 50% of each of your parents. But did you know you also inherit some of their
experiences?

Experiences of earlier generations can modify regulatory factors affecting gene expression such that the DNA
sequence itself is not changed but the individual’s physiology and behavior are substantially influenced.[13]

Instinct and predispositions:

An animal mind is not born as an empty canvas: Bottlenose dolphins know how to swim and honey bees know
how to dance without ever having learned these skills. Little is known about how animals acquire the instincts
that enable such innate behavior. Instincts are widely held to be ancestral to learned behavior but increasing
evidence is showing that these might be epigenetic features passed through the germ line. [5]

For example, when a mouse has experienced fear of something, changes in DNA methylation and chromatin
structure in neurons of the hippocampus help stabilize long-term changes in neural circuits. These changes help
the mouse to remember what has been learned and support the establishment of new behavioral responses.

Evolutionary changes in epigenetic mechanisms may sculpt a learned behavior into an instinct by decreasing
its dependence on external stimuli in favor of an internally regulated program of neural development. Ther e
is evidence for such epigenetically driven evolutionary changes in behavior. For example, differences in innate
aggression levels between races of honey bees can be attributed to evolutionary changes in brain gene
expression that also control the onset of aggressive behavior when threatened. Another example of this intrinsic
epigenetic regulation is female puberty. Currently a few studies have related epigenetic mechanisms to female
puberty regulation, supporting the notion that the activation of neuroendocrine pubertal components is mediated,
at least in part, by epigenetic mechanisms. [73]

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

THE TRUAGE™
Treatment Framework

Epigenetic Age in Utero

You can actually already have age acceleration when
you are in the womb. As almost every child seems to
age at a different rate, many studies have looked into
the epigenetic age of children. The results have told
us a lot!

For instance,
Some findings have shown that accelerated epigenetic
age when children are in the womb are associated with
higher birth weight and birth length as demonstrated
previously and that this difference persisted up to
approximately 9 months of age. From age 9 months
onwards, these differences continued to attenuate
and eventually reversed for weight, resulting in
approximately 0.6 kg lower weight at age 10 years per
week greater gestational advanced aging. [52]

The amount of touch and coddling you get as a child

can affect your aging rate. We know that coddling can
affect your age acceleration but we aren’t quite sure The Impact To You
what implications that this has yet! [60]

Did your mother smoke or use nicotine products

while she was pregnant with you?
The risk of fast aging in children is about 3 times higher
in mothers who smoked versus the risk in nonsmokers.

Did you or your mother have any pregnancy

complications?
Prenatal adverse environment is associated with
epigenetic age deceleration at birth and hypomethylation
at the hypoxia-responsive EP300 gene. P300 gene
revealed cg19011939 to be differentially methylated
in association with prenatal adversity. This can lead to
increased epigenetic age acceleration. [66]

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THE TRUAGE™
Treatment Framework
The Impact To You

Age of parents?

Parental experiences:
Many studies have linked higher paternal age to reduced
benefits in longevity. [82]

Maternal Age:
Unfortunately, maternal age has been documented to be
a big influence on several epigenetic changes.

One of the well-described changes occurs in a gene

called a carbohydrate N-acetylgalactosamine 4-O
sulfotransferase 8 (CHST8). This has some important
implications for fertility. CHST8 is predicted to be
maternally imprinted and encodes an enzyme necessary
for the synthesis of luteinizing hormone (LH).

An LH surge is responsible for triggering ovulation and development of the corpus luteum. This finding may
provide additional support for the hypothesis that increased maternal age results in decreased fertility in adult
daughters via epigenetic modification of critical target genes.

LHX8 is one of 2 genes which are related to maternal age and obesity as well. In addition to LXH8’s key role in
reproduction, is an expression marker for metabolically active brown fat. The other gene linked to brown fat is
PRDM16.

Brown fat mass and BMI have been reported to have an inverse association in adults. Meaning the more brown
fat you have the lower your BMI.

Additionally, we find an inverse association between maternal age and the adult daughter BMI – a relationship
that we confirmed in the full Sister Study cohort. Meaning that the older mom you have the more likely you are
to have a higher BMI.

While the associations between maternal age and offspring cardiometabolic health may be modified by many
other factors, the biologic underpinning for these relationships may include epigenetic modifications at LHX8
and PRDM16. [61]

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THE TRUAGE™
Treatment Framework
The Impact To You

Socioeconomics of your parents at birth

Lower SES was associated with higher methylation
age for children at birth. SES was negatively and
significantly associated with methylation age at birth.
[8]

Childhood events:

Did you have experience stress as a child such

as abuse, financial stress or a parent with a
mental illness?
It seems that there are times throughout development
where we are particularly sensitive to different stimuli.
There are several examples of this.

One study found evidence for sensitive periods during early and middle childhood, when the association between
adversity exposure and epigenetic aging appears to be particularly strong. This finding aligns with human studies
showing the importance of sensitive periods in epigenetic programming [19, 21, 57].

It seems therefore plausible that the epigenetic age of cells is influenced by environmental inputs in a similar
time-susceptibility manner. The current findings further emphasize the importance of attending to possible
time-dependent effects when studying the effects of adversity on cellular aging, including DNAm and other
cellular-based measures of accelerated aging.

The sex-stratified analyses revealed that adversity could differentially affect epigenetic age acceleration in boys
and girls. Some of these associations were particularly notable; for example, by age 7.5, girls who were exposed
to abuse at age 3.5 were biologically older than their unexposed peers by almost 2 months.

Childhood Abuse [45], financial stress [74], and parental psychopathology [7,45], are all associated with accelerated
epigenetic aging in adulthood.

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THE TRUAGE™
Treatment Framework
The Impact To You

Semi-Centenarians: Anyone over 100 in your family?

It isn’t just bad behavior that is passed through
genes. Good behavior and results can also heritable.
Semi- supercentenarians (subjects who reached an age
of 105-109 years) arguably represent the gold standard
of successful human aging because they managed
to avoid or postpone the onset of major age-related
diseases. If you have had one of these individuals in
your family, you are more likely to age much slower!

Further, the offspring of centenarians age more slowly

than age matched controls according to Age Accel and
intrinsic age acceleration. [33]

Infectious inheritance

In April of 2020, a study came out showing the first

example of an infection epigenetic inheritance in men.
This study from Walter and Eliza Hall Institute in Australia
showed an infection of toxoplasmosis in males can
result in epigenetic changes being transmitted to
subsequent generations. [79]

Toxoplasma is one of the world’s most common

parasites, estimated to be carried by between 25 and 80
percent of the global population. Toxoplasma infection
can cause an initial mild illness in most people, however,
pregnant women, babies and people with weakened
immunity experience more severe infections.

The published study documented that Toxoplasma

infection in male mice caused changes in levels of ‘small
RNA’ molecules contained in their sperm, potentially
altering gene expression in the resulting offspring
which could affect development and behavior. Even
viral exposure in previous generations could affect your
epigenetic age. [79]

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

THE TRUAGE™
Treatment Framework
Your Selected Ethnicity

The Impact of Sex, Race, and other demographics

What best describes your ancestry/ethnicity?

Middle Eastern/North African
Race seems to have a significant impact on epigenetics.
We are unsure at the moment how much this affects
different health outcomes but studies have shown the
following:

- Infants from mixed race/ethnicity origin had significantly higher methylation age and higher frequency of fast
aging rate than that in African-origin black people. [38]

- African Americans have indications of a significantly younger immune system age than Caucasians after
controlling for gender, educational level, diabetes status, and Hypertension. [35]

- According to measures of extrinsic epigenetic age acceleration, Hispanics have a significantly older extrinsic
epigenetic age than Caucasians and fewer naïve CD4+ T cells.

- This pattern of fewer naïve CD4+ T cells is even more pronounced for Tsimane, who experience repeated acute
infections and elevated, often chronic, inflammatory loads. [35]

- In one famous study, there were three variables linked to extrinsic epigenetic age acceleration: race/ethnicity,
hypertension, and gender. However, this significant association between extrinsic epigenetic age acceleration and
hypertension, type II diabetes status is only found in Caucasians; not in African Americans. [35]

- The lower level of intrinsic epigenetic age acceleration in Hispanics echo the finding that Hispanics in the US
have a lower overall risk of mortality than Caucasians despite having a disadvantaged risk profile. The fact that
Hispanics have typically had lower intrinsic epigenetic aging but not lower extrinsic epigenetic aging might reflect
that Hispanics have higher levels of metabolic/inflammatory risk profiles and that Hispanics have a lower relative
CD4+ T cell percentage than Caucasians. [35]

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THE TRUAGE™
Treatment Framework Cont.
The Impact To You

What is your Biological Sex?

Sex morbidity–mortality paradox

The sex morbidity–mortality paradox was first described

in the 1970s. It refers to the observation that women
have a lower mortality rate compared to men despite
being more likely to suffer from other diseases and
co-morbid conditions. It has always been assumed that
this might be due to behavioral traits such as lifestyle
factors or that they might be less likely to go to a doctor in
order to be diagnosed with a disease or condition. [44]

However, we still see differences in health after accounting for differences in work-related behavior, smoking,
obesity, and other behaviors. We also see this in epigenetic testing.

Although there is only one study which has really dived into the differences between sexes. It showed that
epigenetic aging markers show a large and consistent male-biased vulnerability in multiple tissues (blood, brain,
and saliva) across all racial groups.

Men have higher IEAA and EEAA than women even when controlling for education, diabetes, and hypertension.
[35]

However, this difference wasn’t found in all races. According to the studies evaluation of EEAA, Caucasian men
are epigenetically older than Caucasian women, but there was not a significant difference in other groups such as
African Americans or central African populations.

Despite the inclusion of race, it is still clear that some of the processes which lead to advanced epigenetic aging
are affecting Men more. Additionally, it might have a larger effect on extrinsic aging because Men have fewer
naïve CD4+ T cells than women in three racial/ethnic groups: Caucasians; Tsimane; and African Americans. [35]

Sex also effects aging of the brain

All tissues have different epigenetic aging rates! However, the impact of sex and tissues aging is still seen in
the brain. While sex did not have a significant effect on the epigenetic age of the cerebellum, the study found
that other brain regions from men exhibit a significantly higher age acceleration than those from women. [35]

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THE TRUAGE™
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Outer circle - Extrinsic aging

Environmental exposures such as nutrition, disease,
stress, activity, medications, and drugs can alter DNA
methylation at various stages in your life. These are
considered extrinsic factors and are the focus for any
health conscious individuals because these are the
things you can fix and change! [2]

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THE TRUAGE™
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Environmental Exposures

The Trouble with Testing and the Twin Solution:

When setting up a good scientific experiment, one of the most important things to do is to have very few
variables. By doing this, scientists are able to calculate just how much one independent variable can affect
the outcome they are measuring. Unfortunately, this is often hard to do. It is especially hard to do with the
epigenetics of methylation because so much can change this pattern. That is why most valuable studies are
done with large numbers of people and in similar populations.

It is similarly difficult judging which things in the environment of a person change their epigenetic aging rate.
Even just defining what should be included in the environment can be incredibly difficult. You might consider
pollution, but do you account for humidity? One way to solve this is to look at individuals who do just about
everything in the same fashion except for one of two variables. That is why identical twins can be so helpful!
Identical twins share the same DNA and oftentimes share the exact same environment growing up. It is as close
to a controlled experiment as you can get for such a complicated field of research like epigenetics!

Thankfully, many of these experiments have been done and they tell us a lot about the environmental effects of
aging. [40] In one study looking at two popular clocks, they estimated that 40% of the determination of the aging
rate was due to the factors you can’t control like your DNA or the influences and lives of your parents. However,
this means that 60% is changeable! This means you’re able to greatly affect your own aging!

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Correlations to Toxic Exposures

Oftentimes, methylation epigenetics doesn’t have Environmental exposures are a good example of this.
enough data yet to be a stand alone diagnostic. Below is a list of some environmental toxins which can
However, we are able to help narrow down the scope of help shape the epigenome. If you are concerned you
diagnosis. One of the ways we can do that is by looking have been exposed to these things please let us know
at the correlated changes of the DNA methylation to and we will work to read your DNA and let you know
different types of diseases or exposures. the likelihood of exposure! [56]

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THE TRUAGE™
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Air Pollutants

Everyone knows that due to environmental changes

and increased activity, the air is becoming more polluted!
To objectively measure the amount of pollution in the
air scientists have created a term called PM2.5. PM2.5
refers to atmospheric particulate matter (PM) that
have a diameter of less than 2.5 micrometers, which
is about 3% the diameter of a human hair.

Commonly written as PM2.5, particles in this category are so small that they can only be seen with a microscope.
Since they are so small and light, fine particles tend to stay longer in the air than heavier particles. This in-
creases the chances of humans and animals inhaling them into the bodies. Owing to their minute size, parti-
cles smaller than 2.5 micrometers are able to bypass the nose and throat and penetrate deep into the lungs
and some may even enter the circulatory system.

Studies have found a close link between exposure to fine particles and premature death from heart and
lung disease. Fine particles are also known to trigger or worsen chronic disease such as asthma, heart
attack, bronchitis and other respiratory problems.

A study published in the Journal of American Medicine suggests that long-term exposure to PM2.5 may lead to
plaque deposits in arteries, causing vascular inflammation and a hardening of the arteries which can eventually
lead to heart attack and stroke. Scientists in the study estimated that for every 10 micrograms per cubic meter
(μg/m3) increase in fine particulate air pollution, there is an associated 4%, 6% and 8% increased risk of
all-cause, cardiopulmonary and lung cancer mortality, respectively. [17]

In addition to those health effects, we also see changes in the epigenome and in the
epigenetic age rate!

In a study with almost 600 men from the Northeastern USA enrolled in the Normative Aging Study (NAS),
a 1 μg/m3 increase in one-year PM2.5 exposure was significantly associated with a 6-month increase in
their epigenetic age. [63] In a similar study using 1,777 German participants of the Cooperative Health
Research in the Region of Augsburg (KORA) study, a 0.97 μg/m3 increase in long-term exposure to
PM2.5 was associated with a 0.33-year increase in extrinsic epigenetic age acceleration. [16] In addition
to PM2.5 mass, associations with a particular clock have been observed with specific PM2.5 components such
as ammonium and sulfate. [16]

Pollution and particles are negative for your aging and you can wear a mask to prevent this when traveling.
Particularly, due to pollution’s specific effect on extrinsic epigenetic aging, it might be related to decrease
immune functioning!
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THE TRUAGE™
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Metal and Pesticide Exposures

Two studies have examined associations between

metal exposures and epigenetic aging to date.

A study of urinary cadmium in 40 non-smoking women

from Thailand and a study of blood cobalt and chromium
levels resulting from chronic exposure due to metal
on metal hip replacements found no associations
between any of the metals examined and Epigenetic
age. It seems that this doesn’t have an effect on these
variables. [76]

In a study of three organochlorine pesticides – (4-chlo-

rophenyl)-1,1-dichloroethene (DDE), hexa- chloroben-
zene (HCB), and trans nonachlor (TNC) – in the plasma
of 967 Swedish individuals. All three exposures were
positively associated with larger differences between
chronological and epigenetic ages, with TNC having
the strongest association. [49]

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THE TRUAGE™
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Infectious Exposures

As mentioned earlier with toxoplasmosis, we know that

viruses can have an effect on epigenetics. However,
how do some common viruses affect epigenetic aging?

Of all of the infectious agents studied for their relation-

ship with epigenetic age, Human Immunodeficiency
Virus (HIV) is the most widely examined.

In one study HIV positive individuals were found to

have brain and blood samples that were 7.4-years
and 5.2-years higher, respectively, compared to
controls.

What’s more, is that the load of the virus was associated

with higher aging rates. Adult male cases with
detectable viral load (>35 HIV copies/mL) had a
3.6-year higher methylation age as compared to
adult male cases with an undetectable viral load. [32]

In addition to HIV, H.Pylori (an abnormal bacteria from the gut), and cytomegalovirus (CMV) have both been
linked to changes in epigenetic aging. [80]

In a study of 1509 German adults who had documented H. pylori infections, were all associated with increases
in epigenetic methylation age of 0.4, 0.6. and 1-year, respectively, independent of white blood cell distributions.
[16] In peripheral blood cells from 122 nonagenarians (a person who is from 90 to 99 years old) and 21 young
healthy controls from a sub-cohort of the Finland Vitality 90+ study, epigenetic methylation age was 2.5-years
higher in CMV positive individuals versus those without disease. [41]

The underlying mechanisms connecting infections and DNAm-age have yet to be elucidated, and alterations in
blood cell composition may play an important role, though other mechanisms are needed to explain associations
in non-blood tissues and those that appear to be independent of assessed blood cell proportions.

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THE TRUAGE™
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Psychosocial Exposures

Psychosocial exposures such as stress, adversity, and

socioeconomic status, may also impact epigenetic aging.

In Hugo’s fictional work Les Misérables, an extreme

stressor causes the main character, Jean Valjean,
to undergo accelerated aging, depicted as rapid
whitening of his hair. This dramatic depiction is just
one among innumerable examples—found in literary
works, movies, and folklore legends—of individuals
whose “biological clocks” appear to tick fast in the
face of life adversity. Beyond fiction, however, the
connection between psychosocial stress and rate
of biological aging is also seen in everyday life and
clinical practice.

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Adrenal Stress and Fatigue

Rate your Lifetime stress 1-10? The Impact To You

An important risk factor for accelerated aging and

aging-related diseases is psychological stress.
Although stressors are ubiquitous in nature and
necessary for survival, excessive and chronic stress
has been associated with accelerated cellular
aging and increased risk for aging-related disease
phenotypes, including cardiovascular disease,
immune dysregulation, and late-life neuropsychiatric
disorders. Furthermore, stressors occurring during
sensitive developmental periods, such as childhood
maltreatment, have been linked with later
development of aging-related diseases. Lastly,
stress-related psychiatric disorders, including major
depression and post-traumatic stress disorder
(PTSD), are themselves risk factors for such diseases.

In a 2015 study by Zannas et al, the authors showed that cumulative lifetime stress may accelerate epigenetic
aging. They also hypothesized that these effects could be driven by glucocorticoid-induced (cortisol) epigenetic
changes. Cortisol is the hormone that is upregulated during stress and can cause people to put on weight. [84]

Glucocorticoids, a class of endocrine signaling hormones which includes cortisol, are a component of the
biological response to stress. In particular, 85 of the 353 loci that comprise the epigenetic clock are located
near glucocorticoid receptor elements, and 110 loci showed altered DNA methylation after exposure to
dexamethasone, a glucocorticoid receptor agonist. [84]

Researchers have proposed biological mechanisms that may connect stress to epigenetic alterations and DNA
methylation age/aging in particular. Accordingly, stress-inducing psychosocial exposures are frequently associated
with epigenetic age.

Limiting stress and this hormone might be a good way to avoid advanced epigenetic aging.

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Stress, Trauma, & Post-traumatic Stress Disorder (PTSD)

In a study of 392 adults recruited from urban hospitals,

the relationship of life stress was associated with
higher epigenetic age, an observation which was
more prominent in older participants and those who
of veterans suffer from
experienced minimal childhood mistreatment. In
PTSD or depression
this particular study, however, epigenetic age was
not related to childhood trauma, current stress,
depressive symptoms or PTSD symptoms.

Combat-related trauma and PTSD have been associated

with increased epigenetic age in a few studies of
veterans. In veterans of the Iraq and Afghanistan
conflicts (N = 281) lifetime PTSD was related to
increased epigenetic age. of veterans suffer from
other mental concerns
In 339 middle-aged, trauma-exposed veterans,
hyperarousal PTSD symptoms were associated with
increased epigenetic age. [84]

In a longitudinal study, epigenetic age was determined

in 96 Dutch military personnel deployed to Afghanistan,
from blood draws conducted before and 6 months
post-deployment. Combat-related trauma was sig-
nificantly associated with an increase in epigenetic
age of about 2 years over the course of deployment.

A meta-analysis of 9 cohorts (n = 2186, civilian and

military) found modest associations of accelerated
epigenetic age with exposure to child trauma, and 1 OUT OF 3
with lifetime PTSD severity. [Dhingra et al. 2018] veterans seek help

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THE TRUAGE™
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Childhood Adversity and Trauma

Studies of childhood and adolescent adversity have

been the first to use biological epigenetic aging as a
potential measure of intervention efficacy, or effect
modification by intervenable factors.

Using 399 parent and child pairs from rural Georgia,

USA one study aimed to assess if parental depressive
symptoms at child age 11 forecast epigenetic age
at child age 20, and the potential of an intervention
program, the Strong African American Families
program (SAAF), which aimed to improve supportive
parenting and family relationships, to moderate
observed associations.

Among the control group, elevated parental depressive

symptoms were associated with future increased
epigenetic age in children, but the association was
abolished in those receiving the SAAF intervention.
Similarly, a longitudinal study of 616 African American
youths (16–17 years old at recruitment) in rural Georgia
found an ameliorating effect of a supportive family
environment on the relationship of epigenetic age
to experience of racism. Among youth with a less
supportive family exposure to higher levels of racial
discrimination during early adulthood was associated
with higher epigenetic age. [16]

In a separate study of 101 children (age 6–13 years)

from a primarily low income, highly traumatized
neighborhood populations in Atlanta, Georgia, direct
experience of violence was significantly associated
with increased epigenetic aging, and there was a
suggestive (p < 0.10) association between witnessing
violence and epigenetic aging. [16]

Associations of childhood adversity and trauma with

biological age measures appear consistently adverse,
unless moderated by an ameliorative co-exposure,
whether preexisting or the product of intervention.

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THE TRUAGE™
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Socioeconomic Status and Hardship

The largest study of socioeconomic status (SES) to

date has shown us some interesting findings. Using
a meta-analysis of cohort specific associations, low
SES was associated with a 0.99-year greater epigenetic
aging rate when comparing the highest to lowest SES
categories. [25]

The trajectory of a socioeconomic life course has

consistent effects on epigenetic age.

Two studies of epigenetic age were conducted in

African-American adolescents residing in rural Georgia
and examined the direct or modifying impact of
economic hardship on epigenetic age using samples
collected around the Great Recession.

One study examined the impact of economic hardship

during the 2007–10 economic recession on epigenetic
age in 330 African American adolescents (mean age
16.6 years in 2007). Adolescents exhibited a mean
1.42-year increase in epigenetic age with each as
categorical measures of economic adversity increased.
These findings are mirrored in observations of
increased allostatic load and decreased self-reported
overall health with increased epigenetic age. [10]

A second study noted that while self-control was

associated with several favorable psychological
outcomes (e.g., lower rates of depressive symptoms,
substance use, and aggressive behavior), among low
SES youths better self-control was associated with an
increased biological age of 2.27-years. In contrast,
among less-disadvantaged subjects, better self-
control was associated with a 2.14-year deceleration.
[16]

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THE TRUAGE™
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The Impact To You

Do you have Allergies and Asthma?

“ We know that the prevalence of allergies and

asthma has been increasing over the past decade.
The genome hasn’t changed, but some of the ways

”
that the environment is interacting with our genomes
may have. - Dawn DeNeo, MD, MPH

At mid-childhood (mean age, 7.8 years) in Project

Viva, epigenetic age and age acceleration were
cross-sectionally associated with greater total serum
IgE levels and greater odds of atopic sensitization.

Every 1-year increase in intrinsic epigenetic age

acceleration was associated with a 1.22, 1.17,
and 1.29 greater odds of atopic sensitization and
environmental and food allergen sensitization. [70]

Extrinsic epigenetic age acceleration was also

cross-sectionally associated with current asthma at
mid-childhood. Biological age and age acceleration
at birth and early childhood were not associated with
mid-childhood allergy or asthma.

Because the epigenetic clock might reflect immune

and developmental components of biological aging,
the Project Viva study suggests pathways through
which molecular epigenetic mechanisms of immunity,
development, and maturation can interact along the
age axis and associate with childhood allergy and
asthma by mid-childhood. [70]

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THE TRUAGE™
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The Impact To You

Education Level:

Higher education has been a demographic variable

that has been linked to many positive health
outcomes including reductions in morbidity and
mortality risk.

How is it correlated to Epigenetic aging?

It seems that it has a significant effect on the extrinsic

epigenetic aging rate of a person according to a
2017 study. In this study it was correlated that those
with more education have less advanced aging. This
doesn’t mean that getting an education will reduce
your epigenetic age, it just means that it is correlated
to that outcome with the populations studied.

This study also looks into fitness and nutrition which

we will discuss later! [72]

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THE TRUAGE™
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Nutrition
Mediterranean Diet
Nutrition is always a hard topic to discuss and there
are many differing opinions out there about what Monthly/
Sweets, Red Meat Small Amounts
is the best diet. Some focus on macronutrients
such as carb intake, others focus on the timing of
Dairy, Eggs, Daily to
meals, and there are very vocal advocates on all Poultry Weekly

sides. However, epigenetic testing gives us a very

objective metric to look at how some diets and
Few Times
Fish, Seafood
supplements can affect the aging process! Per Week

The Mediterranean Diet Olive Oil Daily

Servings

The Mediterranean diet, which is considered by UNESCO

as a heritage of humanity, is a well-balanced mix of Vegetables, Daily
Fruit Servings
nutrients, antioxidants, and anti-inflammatory molecules.
This diet has demonstrated favorable effects on Cereals,
Beans,
cardiovascular risk, blood pressure, cancer, inflammation, Legumes, Daily
Nuts, Servings
or frailty status, and it has been observed that it can Seeds

impact methylation of inflammation-related genes in

peripheral blood cells.

The role of the Mediterranean diet in promoting healthy aging has been recently investigated in a new European
project abbreviated NU-AGE for “New dietary strategies addressing the specific needs of elderly population
for an healthy aging in Europe”. The aim of NU-AGE project is to investigate how an intervention based on the
Mediterranean diet, specifically tailored according to the nutritional needs of people over 65 years of age, can
impact on age-related diseases and functional decline, possibly modulating inflammation and its outcomes.

Baseline and after the 1-year intervention, and results achieved so far in the framework of this study have
demonstrated a beneficial effect of the Mediterranean-like diet on global cognition and episodic memory,
osteoporosis, immune function, and cardiovascular health, as well as on the proteasomal proteolysis.

There still remains little evidence on the biomarkers associated with the Mediterranean diet. However, one study
looked at the effect of 100+ participants and the effect of their epigenetic aging.

Their study observed a trend towards the epigenetic rejuvenation of participants after the nutritional intervention.
The effect was statistically significant in the group of Polish females and in subjects who were epigenetically
older at baseline.

Together, these findings suggest that the Mediterranean diet can promote epigenetic rejuvenation but with
country-, sex-, and individual-specific effects, thus highlighting the need for a personalized approach to
nutritional interventions. [30]
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THE TRUAGE™
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Calorie Restriction

Calorie restriction (CR) without malnutrition is the most

studied and robust non-genetic non-pharmacological
experimental intervention for extending healthspan
and lifespan in multiple animal models!

In budding yeast, fruit flies, and worms, CR can

dramatically extend lifespan (2–3 fold). [77] A 20 to
50% reduction in caloric intake, without malnutrition,
in some strains of rats and mice prolongs median
and maximal lifespan up to 50% and prevents or
delays the onset of many chronic diseases, such
as obesity, type 2 diabetes, cancer, nephropathy,
cardiomyopathy, neurodegeneration, and multiple
autoimmune diseases. [62]

Hundreds of preclinical studies have shown that

dietary restriction, by inhibiting key nutrient-sensing
and inflammatory pathways, activates multiple
molecular pathways that promote proteostasis,
genome stability, stress resistance, and stem cell
function. Data collected in non-human primates
indicate that calorie restriction in combination with
diet quality modifications markedly decreases the
incidence of cardiovascular disease, cancer, diabetes,
and attenuates age-related neurodegeneration,
sarcopenia, and auditory loss.

Finally, data from human studies show that calorie

restriction remains the cornerstone in the prevention
and treatment of obesity and its complications.
Moderate CR achieved through intermittent fasting
or restricting feeding in combination with regular
physical activity most likely exerts additional beneficial
health effects even in non-obese individuals. However,
a general facet of modern life in the developed world
is the near-constant availability of food. So some
researchers have developed some diets to mimic
fasting or calorie restriction! [62]

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Dr. Valter Longo’s Fasting Mimicking Diet

One of the most popular calorie-restricting diets is

the Fasting Mimicking Diet (FMD) popularized by Dr.
Valter Longo. The longevity diet Valter Longo developed
consists of two parts: the “fasting” phase and the
“whole foods” phase. The fasting phase isn’t necessarily
complete fasting. Rather, it involves consuming a
series of packaged herbal teas, soup blends, energy
bars, oils, and drinks.

They are to be rationed over the course of five days. After those five days, the dieter can go on to the whole
foods phase. During the whole foods phase, the dieter can go back to eating “normal” food that they can get
at the grocery store. They do have to make healthy eating choices, though, or all the space freed up during the
fasting phase will be put to waste. There’s a wealth of research on how intermittent fasting might help with
longevity and youthfulness. Intermittent fasting, they say, can reset our bodies and our genes. [50]

While there is not yet a trial published on this at the moment, there is a highly popularized anecdotal trial on this
by the radiant Gwenyth Paltrow and her company, Goop Lab. Here Gwenyth and two of her team members test
whether different diets can make your body younger, and therefore healthier, by reducing their epigenetic age.
Gwenyth, 46 years old at the time of filming, was prescribed the most extreme of the test diets, the fasting
mimicking diet. Following this five-day cleanse, her biological age was 44.2. Goop’s chief content officer, Elise
Loehnen, 40 years old, was put on a three-week Mediterranean-style diet. Her biological age was 37.9 after
the pescatarian, mostly plant-based diet. Wendy Lauria, Goop’s vice president of marketing, 49.5 years old, was
assigned a three-week vegan diet of plant-based meals, excluding all animal products like meat, fish, milk, and
eggs. Lauria had a biological age of 48.4 after her given diet.

In conclusion, Gwenyth came out on top in the final test results, which showed that her fast-mimicking regimen
shaved 1.7 years off her biological age, bringing her to a youthful 42.5 (compared with her actual age of 46
years). The results of her experiment have been backed up by some data from mouse clinical trials. A 2017
study from the Max Planck Institute for Biology of Ageing looked at dietary restriction in mice and its effect on
genome wide methylation. While they didn’t directly address epigenetic age, they found that dietary restriction
is generally strongly protective against age-related changes in DNA methylation. During aging with dietary
restriction, DNA methylation becomes targeted to gene bodies and is associated with reduced gene expression,
particularly of genes involved in lipid metabolism.

Overall their results revealed that dietary restriction remodels genome-wide patterns of DNA methylation so
that age-related changes are profoundly delayed, while changes at loci involved in lipid metabolism affect
gene expression and the resulting lipid profile. [31]

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Bioactive Compounds and Longevity

Although calorie restriction has been shown to have

a beneficial role in aging, calorie restriction therapy
has several limitations or potential side effects, such
as infertility, menstrual irregularities, hypertension,
and depression. For these reasons, recent studies
have aimed to identify bioactive compounds that
may mimic calorie restriction and provide therapeutic
anti-aging effects.

Unfortunately, direct evidence shows the linkage

between bioactive compound consumption and
longevity is scarce. However, several studies have
reported beneficial effects of natural compounds on
age-related phenomena, mainly in providing anticancer
and anti-inflammatory effects. [31]

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THE TRUAGE™
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Resveratrol
Resveratrol is the most characterized bioactive
polyphenolic compound in anti-aging diets. Dietary
polyphenols have antioxidant capacity and protect
against age-related degenerative diseases. They can
activate endogenous defense systems and modulate
cellular signaling processes. Resveratrol is found in
grapes, grape-based red wine, as well as strawberries
and blueberries. [67]

The possible role of resveratrol in extending the life

span recently gained worldwide attention. Resveratrol
has been identified as a potent SIRT1 activator that
mimics the effects of calorie restriction and regulates
longevity from lower organisms to humans. In 2003,
Howitz and colleagues showed that resveratrol
increases the deacetylase activity of SIRT. A number
of studies showed that resveratrol induced SIRT1
activity in several species. Resveratrol mimics calorie
restriction effects, which may result in an increased
life span.

Although the effects of resveratrol and SIRT1 on longevity are still debated, resveratrol clearly appears to
improve metabolism and attenuate the risk of age-related chronic diseases in animal models. For example,
increased SIRT1 activation from resveratrol improves energy expenditure and prevents diet-induced obesity
and other metabolic diseases. [36]

In addition, middle-aged mice on a high-calorie diet that were treated with resveratrol showed health and
longevity benefits. In humans, resveratrol supplementation induces metabolic changes in obese humans,
mimicking the effects of calorie restriction. In addition to metabolic regulation, resveratrol has an intrinsic
antioxidant capacity and induces the expression of antioxidant enzymes, which reduces oxidative stress.
To date, little is known about the underlying epigenetic mechanism by which resveratrol improves longevity
and aging-related metabolism. Research suggests that resveratrol may target metabolism-related pathways,
such as AMP-activated protein kinase and peroxisome proliferator-activated receptor-gamma coactivator 1 a. [6]

In addition to resveratrol, you may see other research on several bioactive components of which beneficial
effects are mediated by epigenetic modifications, namely, sulforaphane, epigallocatechin-3-gallate (EGCG),
quercetin, and genistein. All of these have been highlighted for their epigenetic effect on aging. [69]

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THE TRUAGE™
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Eating Fish and Chicken

In the same study that looked at the dietary effects
of alcohol, they also looked at how the consumption
of fish and chicken affected epigenetic aging. Those
who had fish more frequently were less likely to have
a higher extrinsic epigenetic age acceleration. This is
consistent with prospective studies suggesting that
fish consumption is protective against various age-
related diseases. The benefits of fish intake may be
mediated in part through the omega-3 fatty acids, Extrinsic Intrinsic
eicosapentaenoic acid (EPA) and docosahexaenoic Epigenetic Age Epigenetic Age
acid (DHA), which stimulate the synthesis of anti-
Fish Poultry
inflammatory cytokines. This was further supported
by the authors’ findings that CRP—a well-known Fruits & Veggies HDL Cholesterol
marker of inflammation—was the most significant
explanatory biomarker of extrinsic epigenetic age Moderate Alcohol Insulin & Glucose
acceleration. Edication & Income C-Reactive Protein

This suggests that one reason higher fish consumption Exercise BMI & Waist-to-Hip Ratio
may lower extrinsic epigenetic age acceleration is HDL Cholesterol Triglycerides
because it has beneficial anti-inflammatory or
Insulin & Glucose Systolic Blood Pressure
metabolic effects.
C-Reactive Protein
Similarly, the same study found that poultry
BMI & Waist-to-Hip Ratio
consumption was associated with a decreased
intrinsic age rate and lower BMI after adjusting for Triglycerides
potential confounders. [72]
Systolic Blood Pressure

Blue Arrow: Decreases Epigenetic Age

Red Arrow: Increases Epigenetic Age

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THE TRUAGE™
Treatment Framework

Alcohol
While many might instinctively think that alcohol might
decrease epigenetic age, the one study conducted on
this actually showed the opposite!

They found that alcohol consumption was negatively

associated with extrinsic epigenetic age acceleration
even after adjusting for potential confounders such
as socioeconomic status! This is consistent with
prospective studies which have identified light to
moderate alcohol intake as a protective factor against
all-cause and CHD-related mortality. It is even supported
by a recent publication that also found an inverse
association between epigenetic age and alcohol
intake in Caucasian and African American individuals.

In a study from UCLA, researchers found that one

monthly drink had a positive effect. It remained
consistent when adding weekly and daily intake
levels as well.

The association appears to be driven by wine

consumption though there is also a trend towards
association with beer. This is consistent with other
studies that have suggested that wine may have
added benefits compared to light alcohol consumption.

It has been postulated that this may also be related

to the anti-inflammatory effects of light alcohol
consumption, which are associated with decreased
circulating levels of inflammatory markers such as
IL-6 and CRP. [72]

Alternatively, this may be the result of reverse

causation, whereby those with health issues abstain
from alcohol consumption due to their illness,
however, other interventional studies support a
protective effect of moderate alcohol consumption.

Do not change drinking habits without talking to

your physician.

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THE TRUAGE™
Treatment Framework

What is your alcohol consumption?

Some studies which have been published have found

that alcohol consumption can help reduce your aging
rate. Particularly, it can help reduce your extrinsic
epigenetic aging rate even after adjusting for potential
confounders such as socioeconomic status. Do you smoke?
Tobacco
This finding is consistent with prospective studies
which have identified light to moderate alcohol Tobacco smoking is a major public health problem,
intake as a protective factor against all-cause and CHD- associated with substantial preventable morbidity
related mortality and are supported by a recent globally. In particular, active smoking in adults accounts
publication that also found an inverse association for a large proportion of age-related diseases, including
between epigenetic age and alcohol intake in Caucasian various forms of cancer, respiratory, and cardiovascular
and African American individuals diseases. Recent studies have demonstrated the role
of DNA methylation, one of the main forms of epigenetic
In the same study, they also found that the potential modification, in the pathways of smoking and smoking-
benefits of alcohol consumption are observed using induced diseases via regulating gene expression and
a threshold of more than one serving per month. But genome stability.
the effect is extended to a drink once weekly and once
daily. The association appears to be driven by wine An increasing number of smoking-related CpG sites
consumption though there is also a trend towards in various genes, such as AHRR, F2RL3, and GPR15,
association with beer. This is consistent with other have been discovered by epigenome-wide association
studies that have suggested that wine may have added studies (EWASs) based on whole blood samples, and
benefits compared to light alcohol consumption. have been shown to be useful as quantitative biomarkers
of current and past smoking exposure and predictors
This finding may also be related to the anti-inflammatory of smoking-associated health risks. This means you
effects of light alcohol consumption, associated with might be able to have a blood test in the future which
decreased circulating levels of inflammatory markers quantifies how much you have smoked in life!
such as IL-6 and C-Reactive Protein (CRP). [72]
Despite many changes in the epigenome, smoking
isn’t necessarily correlated to increased epigenetic
age which highlights that not every poor lifestyle
The Impact To You choice is associated with an increased epigenetic
aging effect in blood tissue. [29]

Illicit Drugs
Not many studies have yet been done on illicit drugs,
however, there are many in development!

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THE TRUAGE™
Treatment Framework

Pregnancy

Have you been pregnant?

Pregnancy also affects aging. One study suggests that
Pregnancy (gravidity) predicts shorter telomeres and
epigenetic age acceleration, measures of mitotic and
non-mitotic aging, respectively, among young women.

Unfortunately, this supports other data showing the

cost of reproduction from pregnancy in humans as it
relates to age. [75]

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THE TRUAGE™
Treatment Framework

Diseases
CHD

DNA methylation is associated with the risk of developing

coronary heart disease (CHD), using methylation levels
at 52 CpG sites determines one’s risk of developing
CHD. CHD events include unstable angina, heart
attack, coronary revascularization, and coronary death.

Your race/ethnicity, chronological age, and sex are related to your susceptibility to developing CHD. Biomarkers
of epigenetic aging can address the mortality rates of coronary heart disease and how epigenetic rates of aging
are found to be significantly associated with race, sex, and chronological age.

In the first-ever study to use epigenetic measures as an estimate for aging rates amongst gender and ethnic
groups found differential mortality rates across these groups. The study used thousands of participants across
these ethnic groups: 1387 African ancestry, 2932 caucasian, 657 Hispanic, 127 east Asians, and 59 Tsimane
Amerindians.

Finding that women have lower rates of mortality than men despite having higher rates of morbidity.
Hispanics and Tsimane have lower intrinsic epigenetic age acceleration (IEAA) and longer life expectancies
but higher extrinsic epigenetic aging rates than Caucasians. African Americans have lower extrinsic
epigenetic age acceleration (EEAA) than Caucasians and Hispanics.

Notably IEAA is not associated with CHD risk factors, but EEAA was positively correlated with CHD risk factors
like triglyceride and creatinine levels. [35]

Large-scale studies show that risk factors for CHD include smoking, obesity, hypertension, serum lipids, and
type-2 diabetes are all linked to differences in leukocyte DNA methylation. The largest longitudinal study of its
kind, with 11,461 participants found pathways to CHD, including calcium regulation, kidney function, and gene
regulation mechanisms that involve non-coding RNAs.

Associations between leukocyte DNA methylation and risk of CHD are clinically relevant. These associations
have the potential to present novel avenues for targeting disease pathways and developing therapeutic
interventions. Several of the 52 CHD associated CpG sites map to genes with roles in calcium regulation and
kidney function. [1]

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THE TRUAGE™
Treatment Framework

Downs Syndrome

Individuals with the genetic disorder, Down syndrome,

have an increased risk of having chronic diseases
that are associated with older age. The disorder is
interpreted as segmental progeria affecting at least
two different tissues and is characterized by rapid
aging starting in early childhood.

Not only is this an intellectual disability but it is also

linked to the clinical manifestations of rapid aging.
Trisomy 21 is linked to the acceleration of the biological
age of tissues and significantly increases the ages of
blood and brain tissue.

A method that predicts adults with Down’s syndrome’s

biological age is based on DNA methylation at 353
CpG sites. These sites are strong indicators for these
individuals’ biological age. Using CpGs associated with
Downs syndrome status and age acceleration helps
determine the specific set of conditions associated
with accelerated aging.

The early onset of epigenetic changes is linked

to Downs syndrome pathologies which include
premature wrinkling, greying of hair, hypogonadism,
early menopause, hypothyroidism, declining immune
function, and Alzheimer’s disease. [34]

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THE TRUAGE™
Treatment Framework

Autism

The prevalence of autism spectrum disorder has been

increasing over the past 20 years. Autism spectrum
disorder is a clinical grouping of neurodevelopmental
disorders characterized by debilitated social commu-
nication and repetitive behaviors. Many children and
adults diagnosed with autism have comorbid health
problems and are three to ten times more likely to die
prematurely. The biological makeup of people with
autism spectrum disorder is linked to other illnesses
such as epilepsy, gastrointestinal, and respiratory
disorders. [68]

“ Comorbidity is to be expected in autism spectrum

disorders- directly or indirectly. Comorbid conditions
may be markers for underlying pathophysiology and

”
request a more varied treatment approach.

-Jorn Isaksen et al., 2012 ‘Children with autism spectrum

disorders: The importance of medical investigations.’

Genetic discoveries of autism spectrum disorder provide evidence of a strong inherent component for many
cases of individuals with the disorder. Multiple genetic hits for autism spectrum disorders located in numerous
genes support that genetic risk for autism spectrum disorder will likely lie in immune-related genes. But genetic
inheritance is not the only cause for the pathology of the disorder.

Impairments of microglial function explain the mechanisms that react to environmental influences on the
developing brain’s DNA methylation. Individuals with autism spectrum disorders exhibit age-related changes
in the trajectory of microglial and synaptic function, suggesting a genetic risk for autism can influence regional
cortical gene expression.

Epigenetic dysregulation of synaptic genes at the transcriptional level contributes to autism susceptibility.
Abnormal epigenetic modifications, known as epimutations in DNA, can be acquired throughout life. Impaired
methylation is evident in environmental factors’ role in autism risk. High levels of impaired methylation are
common in people affected by autism compared to other groups and it has a pathological role in the development
of autism spectrum disorder. [58]

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THE TRUAGE™
Treatment Framework

Osteoporosis

Osteoporosis is an age-related progressive bone disease

characterized by the reduced bone formation and
the accumulation of adipocytes in the bone marrow
compartment, causing the bones to become porous
and weak. Epigenetic mechanisms play a significant
role in the activity of bone cells.

Diseased patients suffer from changes in the way their

DNA is methylated, causing the acceleration of the
disease. Those who have an accelerated epigenetic
age are more likely to experience the disease, notably,
age is the most important risk factor for osteoporosis.

Adipocytes are derived from mesenchymal stem cells,

these stem cells are important for making and repairing
skeletal tissue. At the expense of bone formation,
osteoporosisaberrantlineageallocationofmesenchymal
stem cells leads to the overwhelming accumulation
of marrow adipose tissue. The over-accumulation of
marrow adipose tissue occurs in states of low bone
density and can be harmful to the overall health of the
diseased individual.

Marrow adipose tissue influences mesenchymal stem cell lineage decisions, the bone responds to various
environmental cues during aging. Epigenetic regulation of mesenchymal stem cell lineage specification plays a
role in osteoporosis, resulting in a mesenchymal stem cell shift from osteolineage to adipocytes. These changes
can lead to a bone matrix that becomes thin and porous.[83]

Bone tissue samples from patients with osteoporosis and healthy patients uncovered inhibitors of bone formation
with methylation levels being significantly different between osteoporotic and control patients. Epigenetic
events may have a profound effect on the differentiation and activity of the cells within the bone marrow
environment and consequently may contribute to the pathophysiology of age-related bone loss. [46]

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THE TRUAGE™
Treatment Framework

Hypertension

Hypertension is a complex condition with no single

causative agent, remaining one of the world’s leading
health problems. It is high blood pressure
characterized by the long-term force of the blood
against the artery walls able to cause major health
problems, such as heart disease.

Evidence supports that epigenetic modifications are

just as important as any genetic predisposition for
the development of hypertension. The interaction
between genetic and environmental systems can
determine an individual’s risk for hypertension.

Different degrees of DNA methylation has been

correlated with the onset, timing, and severity of
hypertension. Age acceleration in terms of the
differences between age predicted by DNA methylation
and chronological age is an independent predictor
of all-cause and cause-specific mortality in patients
with hypertension.

Global genomic DNA methylation can be quantified by measuring the amount of the 5-methyl cytosines
present in a DNA sample. A study found a correlation between the decreased levels of 5-methylcytosine in
peripheral blood with an increase in hypertension grade severity. Global DNA methylation levels decreased as
the severity of hypertension increased [81].

The most sound data involving the methylation in blood pressure regulation performed a genome-wide
association and replication study. Their results show the roles of DNA methylation in blood pressure regulation.
They identified genetic variants at 12 new loci that correlated with blood pressure modulation in 320,251
individuals of East Asian, European, and South Asian ancestry. At some of the loci, they identified DNA
methylation may lie on the regulatory pathway linking sequence variation to blood pressure [42].

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THE TRUAGE™
Treatment Framework

Diabetes Mellitus

Type 2 diabetes is characterized by chronic

hyperglycemia due to impaired insulin secretion. As a
result of a worldwide aging population and increasing
prevalence of obesity, the number of patients with
type 2 diabetes has rapidly increased. Genome-wide
association studies have shown that an individual’s
genetic background can influence the risk of this
disease.

Epigenetics may affect the pathogenesis of type 2

diabetes. To determine the epigenetic basis of type-2
diabetes a study analyzed DNA methylation at
479,927 CpG sites in human pancreatic islets, which
are regions of the pancreas that produce endocrine
cells, from 15 type 2 diabetic donors and 34
non-diabetic donors.

They ran an analysis to find absolute differences in DNA methylation that was greater than 5%. 1,649 CpG
sites had absolute differences in methylation between the diabetic and non-diabetic islets. Of these CpG sites
97% showed a decrease in DNA methylation within the diabetic islets compared to the non -diabetic islets.
The majority of the CpG sites that showed decreased DNA methylation in the diabetic islets had an intermediate
degree of methylation, with 20-70% being methylated, and were more dynamic to change in human islets.

The study also found that islet expression involved in de novo DNA methylation correlated negatively with
age. Age was associated with differential DNA methylation of 28 CpG sites and ~92% of CpG sites exhibited
differential DNA methylation due to increasing age. These findings suggest that increased aging affects DNA
methylation of CpG sites in the diabetic islets. [14]

It has been suggested that epigenetic changes can contribute to the occurrence of comorbid diseases.
Another study found that epigenetic changes, especially after stress, can be of importance in the pathogenesis
of both type 2 diabetes and depression.

Alterations in gene expression were found in postmortem specimens from a person with type 2 diabetes
compared to controls without diabetes. There is a possibility for epigenetic mechanisms to explain the
increased risk of dementia among individuals with diabetes. Epigenetic mechanisms explain the increased
prevalence and incidence of depression among persons with diabetes as well [3].

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THE TRUAGE™
Treatment Framework

Schizophrenia

The majority of deaths in schizophrenic patients have

been attributed to age-related diseases that are primarily
independent of the brain, such as cardiovascular and
respiratory diseases. Due to accelerated biological
aging in the schizophrenic population, there is an
increased prevalence of these age-related disabilities
and morbidities.

A study on epigenetic aging in blood affected by schizophrenia found that the change in age is significantly
altered by this severe mental disorder. They used a novel blood-based DNA methylation test to be a strong
predictor of morbidity and mortality, it found that epigenetic age is accelerated in late adulthood for
schizophrenic individuals.

Based on the biological age indicated by their genome-wide DNA methylation, schizophrenic patients were
on average 1.55 years older than their chronological age.

Surprisingly they found that individuals diagnosed with schizophrenia displayed epigenetic age deceleration
in young and mid-adulthood. Between these age groups of young and middle-aged adults, schizophrenic
patients were on average 0.7 years younger in their biological age indicated by the level of methylation within
their DNA.

Age-specific effects in schizophrenia can yield new insights that might otherwise be missed. Blood-based
epigenetic aging is a heritable trait and a predictor of a wide variety of phenotypes for this population of
individuals vulnerable to age-related diseases and excess mortality [65]. Understanding the epigenetic
mechanisms that occur between the period of middle adulthood and late adulthood is pertinent to combatting
the acceleration of aging in schizophrenic people.

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THE TRUAGE™
Treatment Framework

Insomnia

Insomnia is a common sleep disorder that makes it

hard to fall asleep and stay asleep; its symptoms are
connected to a multitude of age-related conditions.
Accelerated biological aging may be a mechanism
through which sleep influences risk for age-related
disease and early mortality.

Insomnia symptoms are associated with an increased

vulnerability to declines in mental and physical health,
elevated inflammation, and age-related morbidity and
mortality including risk for coronary heart disease and
cancer. Cross-sectional data suggests that insomnia
is linked to shorter leukocyte telomere length, which
is a biomarker of aging and predictor of age-related
disease risk.

Another biomarker for aging is based on DNA

methylation. Using DNA methylation as a biomarker
of age can examine the role of environmental factors
contributing to the accelerated rate of aging and sleep
disturbances.

Extrinsic epigenetic age acceleration is the deviation

between DNA methylation and chronological age.
Insomnia symptoms are significantly associated with
the extrinsic measure of age acceleration.

Sleep duration and disturbances are associated with

increased epigenetic age of blood tissue and high-
er counts of late CD8+ T cells. These findings link
insomnia with accelerated epigenetic aging and are
evidence of an aged immune system. The importance
of aging biology for late-life chronic diseases, there is
a growing demand to address the sleep disturbance.
[9]

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THE TRUAGE™
Treatment Framework

Dementia

The current public health priority, dementia, is

distinguished by the structural decline in the brain.
Due to the disease’s wide list of symptoms, cognitive
and behavioral tests don’t provide a definitive
diagnosis of dementia which addresses the importance
of biomarkers able to indicate the disease state.

Age-related changes in brain structure are the focus

of dementia research. Increasing age is significantly
associated with the increasing prevalence and
incidence rates of dementia. Finding the link between
age-acceleration propagated by DNA methylation
and the onset of dementia is significant for targeting
therapies that can prevent the disease.

Epigenetic disruptions in the brain are observed in individuals with dementia. DNA methylation markers can
reflect the biological processes that occur in the early stages of dementia. DNA methylation changes have been
observed in individuals with dementia. [28]

Epigenetic evidence suggests that dementia is not a suddenly occurring and sharply delineated state, but
rather it is a gradual change of crucial cellular pathways going into a dysfunctional state. Locating the physical
pathway for gene-environment interactions that lead to dementia is vital.

A case using two monozygotic twins found they had very different DNA methylation in brain cortical neurons.
The twin with dementia had lower methylation than the healthy twin. Reduced DNA methylation is an overall
trend in brain samples of dementia patients compared to people without the disease.

Evidence suggests that epigenetics can detect, prevent, and reverse such processes before clinical dementia is
detected. A study found that individual epigenetic variation is not time-bound and that epigenome differences
correspond to environmental influences, such as smoking. Likewise, DNA methylation changes that occur in
response to stress often occur later in life. [54]

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THE TRUAGE™
Treatment Framework

Alzheimer’s Disease

Aging is tied to a number of neurodegenerative

diseases that cause cognitive decline. Alzheimer’s
disease is the most common form of dementia,
accounting for ~70% of all dementia cases. After the
age of 65 the risk for developing Alzheimer’s disease
doubles every 5 years. The pathogenesis of Alzheimer’s
disease involves gene-environment interactions that
can be captured by the epigenome. Patterns of DNA
methylation are investigated to uncover its relationship
with Alzheimer’s disease.

DNA methylation levels are used to measure the age

of human tissues; also its variant levels of methylation
are linked to the pathology of Alzheimer’s disease.
Aging is associated with the rapidly increasing
susceptibility to Alzheimer’s disease. Using DNA
methylation as an indicator for the rate of disease
progression we can understand that people
diagnosed with Alzheimer’s disease will have a higher
DNA methylation age than their chronological age.

Epigenetic age acceleration, which is the change between epigenetic age and chronological age, captures
the biological age of brain tissue. Alzheimer’s disease patients have biologically older brains than non-
diseased individuals.

In a recent study, age indicated by methylated DNA is associated with cognitive decline among people with
Alzheimer’s disease. Variance in global cognitive function and episodic memory change is much higher in
individuals with Alzheimer’s disease than those without it. It has also been determined in this study that
individuals with Alzheimer’s disease experience far more cognitive aging changes than non-diseased individuals.

Alzheimer’s disease is currently not treatable once patients are diagnosed, but it is definitely a preventable
disease. Dietary supplements and lifestyle changes are the best measures to take when combatting
Alzheimer’s disease. Understanding the mechanisms that drive the acceleration of aging which contributes
to the symptoms of Alzheimer’s disease is pertinent when addressing the treatment and prevention of this
disease. [48]

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THE TRUAGE™
Treatment Framework

Cancer Risk

As we have mentioned, the epigenetic aging rates of all

tissues are different. This is true with cancer as well as
epigenetic age does not always parallel chronological
age, particularly in tumor samples.

This is often referred to as biological age or the EpiAge™

ratio. Furthermore, since the methods for measuring
epigenetic age incorporate loci in pathways related
to both cancer development and aging in general
(e.g., DNA damage, cellular proliferation, and oxidative
stress), it is highly possible that biological age can be
a predictive biomarker for cancer risk, metastasis, and
mortality in addition to serving as an indicator of aging.

With further study and refinement, the concept of epigenetic age may also be useful for improving our
understanding of mechanisms by which age and cancer are related. However, no longitudinal analysis has yet
evaluated how blood epigenetic age changes overtime prior to cancer diagnosis or cancer-related death, and
whether blood biological age can predict future risk of cancer incidence and mortality.

In one study though, they investigated patients’ epigenetic markers and their cancer progression. What they
found was interesting. About 3–5 years before cancer onset or death, biological age was associated with
cancer risks in a dose-responsive manner and a one-year increase in biological age was associated with
increased cancer incidence and mortality.

Participants with smaller biological age and decelerated epigenetic aging over time had the lowest risks of
cancer incidence (P = 0.003) and mortality (P = 0.02).

This isn’t enough to have a conclusive link for prediction but it was concluded that blood epigenetic age may
mirror epigenetic abnormalities related to cancer development, and might potentially serve as a minimally
invasive biomarker for cancer early detection. [85]

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THE TRUAGE™
Treatment Framework
The Impact To You

Have you been through menopause and do you

have AA?

Breast cancer’s link:

Increased breast cancer risk has been correlated to

increased epigenetic aging rate in women who are
postmenopausal (particularly intrinsic epigenetic
aging rate) in a 2017 study. Another reason to try and
slow your aging rate. [20]

Do you have a history of lung cancer?

Lung Cancer incidence

A separate study by Levine et al in 2015 also showed
a correlation between lung cancer risk and intrinsic
epigenetic aging. [47]

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THE TRUAGE™
Treatment Framework
The Impact To You

Medications

GH, DHEA, Metformin (The Famous TRIIM Study)

Are you taking Growth Hormone or a Growth

Hormone secretagogue?

In mammals, the thymus supports the maturation of

thymocytes into T-cells and to maintain immunological
tolerance. This is an important part of what makes
your immune system work.

The thymus continually atrophies after childhood, it’s

epithelia is replaced by adipose tissue, and it has been
hypothesized that at least some immune dysfunction
associated with aging is caused by the loss of
thymus tissue. For example, infants whose thymus
was removed during cardiac surgery at 18 years after
surgery have immune cell populations more reminiscent
of 65-70 year olds.

Multiple factors have been tested preclinically to

try and regenerate the Thymus. However, only one
study has been shown to help regrow the thymus for
immune benefit. [39]

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THE TRUAGE™
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THE FIRST HUMAN CLINICAL TRIAL DESIGNED TO REDUCE EPIGENETIC AGE

In what may be the first human clinical trial designed to reverse aspects of human aging, the TRIIM (Thymus
Regeneration, Immunorestoration, and Insulin Mitigation) study was conducted to address the thymus
regeneration and therefore help with epigenetic aging! The decision to use GH, DHEA, and metformin.

The scientific justification for using GH to regrow thymic tissue when it has not completely atrophied is extensive,
as the thymic epithelia not only express GH receptors but secrete GH in a positive feedback loop.

Fahy et al. treated 9 subjects in this uncontrolled Phase I trial with GH. Because GH induces hyperinsulinemia,
GH was supplemented with metformin, an AMPK activator that increases glucose tolerance and DHEA, an
endogenous steroid hormone that decreases gradually in adults, that can act as a precursor to testosterone,
estrogen, as a neurosteroid that Fahy has stated to have anti-diabetic properties, but there is little reported
evidence for this.

After treatment of 9 patients for 12 months, the thymic fat-free fraction (TFFF) (a measure of functional thymus
tissue) increased significantly in 7 out of 9 participants, in many cases almost doubling from 20% to ~35%. The
two nonresponders were actually participants who had the highest TFFF at the start of the trial. These results
were considered statistically significant but suffer from the underpowered number of participants.

The most significant immune changes observed were decreases in total and CD38+ monocytes which were
statistically correlated with the TFFF. The reduction led to an increase in the lymphocyte-to-monocyte ratio (LMR),
which reached ratios similar to younger adults. These changes are consistent with restored thymic function.

Because GH acts predominantly through induction of IGF-1, Fahy et al. wanted to ensure that they did not accelerate
aging. Currently, various DNAm age clocks are the best correlated biomarkers available for chronological age,
phenotypic age and mortality. To their surprise, DNA age using leukocytes at 12 months of treatment was
reversed in all four clocks studied with a mean change using regression on all 4 clocks of -2.5 years. These
included the Horvath clock[9] which can predict chronological age for a large number of tissues (-2.5 years), the
Hannum clock (- 3 years) which is based on leukocytes in whole blood, the phenoage DNAm clock (-3.5 years),
which is better correlated with aging phenotype and the GrimAge DNAm clock (-2 years) which predicts human
life expectancy. After cessation of treatment all of the clocks resumed ticking, except the Grimage clock which
did not advance. The rates of decline increased in the last 9-12 month period which could indicate further gains
are possible with longer treatment

It is interesting that two of the three factors decrease with age (GH and DHEA) as might be expected for factors
that help maintain homeostasis! It also might give some credence to hormone replacement therapies. [23]

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THE TRUAGE™
Treatment Framework
The Impact To You

Are you taking Metformin?

Metformin, an FDA approved first-line drug for the

treatment of type 2 diabetes, has known beneficial
effects on glucose metabolism.

Evidence from animal models and in vitro studies

suggest that in addition to its effects on glucose
metabolism, metformin may influence metabolic and
cellular processes associated with the development of
age-related conditions, such as inflammation, oxidative
damage, diminished autophagy, cell senescence,
and apoptosis. As such, metformin is of particular
interest in clinical translational research in aging
since it may influence fundamental aging factors that
underlie multiple age-related conditions. [4] Also, in
the hallmark Fahy trial, it was part of the drug cocktail
which was wildly successful in reducing the epigenetic
aging rate. [23]

So does Metformin have an effect on epigenetic aging?

Unfortunately the answer is probably not.

In one study, no significant differences were found

for the two metformin groups, and in most cases
those currently on Metformin had a somewhat higher
epigenetic aging rate than those who would be
prescribed Metformin in the future.

Also, the study’s results for the extrinsic epigenetic

aging rate show very little difference between those
who start Metformin between first and second blood
draw and those who don’t. [72]

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THE TRUAGE™
Treatment Framework

Death

No one likes to talk about death…but it is an important

outcome metric when considering your health and
preventative health. This is also an outcome that many
have focused on in the field of Epigenetic aging.

There are multiple studies that link aging rates and

the risk of death. Soon, TruDiagnostic™ will have a
mortality predictor to help determine your risk. In the
meantime, we have summarized some of the findings
from the literature below!

General correlations:
“Increased Age Acceleration has been associated with increased risk of mortality, and these associations,
albeit small, were independent of known mortality risk factors, including a large number of demographic,
lifestyle, and anthropometric variables, and medical conditions.” [18]

Both intrinsic and extrinsic measures of epigenetic age acceleration in the blood are associated with an
increased risk of death from all-natural causes even after accounting for known risk factors.(111)

The different types of tests:

There are many algorithms that can help you predict death risk. The good news is that almost all measures
are pretty good at predicting life expectancy.

A meta-analysis of blood DNA methylation data from more than 13,000 individuals found that all measures of
age acceleration considered were able to predict life expectancy.

Intrinsic versus extrinsic:

Generally, intrinsic measures of epigenetic age exhibit much weaker associations with lifestyle factors and
markers of inflammation, which probably reflects the fact that it relates to an innate aging process that doesn’t
capture other comorbidities. As a result, extrinsic age tends to be more predictive of death.

The measure of extrinsic age acceleration also reflects aspects of immunosenescence. This is because, by
construction, it correlates with age-related changes in blood cell composition, such as T lymphocyte populations,
which underlie much of the age-related decline in the protective immune response. [22]

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THE TRUAGE™
Treatment Framework

It also means that the highly predictive significance of

EEAA for all-cause mortality probably reflects the fact
that it assesses multiple aspects of the biological age
of the immune system including both changes in the
blood cells.

This data also suggests that intrinsic epigenetic age

acceleration is reflective of an intrinsic epigenetic
clock that is associated with mortality independent of
chronological age, changes in blood cell composition,
and traditional risk factors of mortality.

This means that it also is probably indicative of a process

of aging not related to cell composition and is still useful.
For instance, IEAA but not EEAA is predictive of lung
cancer. [47]

Additionally, IEAA is also related to centenarian status.

[33]

Methylation + Biomarkers = better death prediction capabilities:

Methods of biological testing which use biological methylation markers and other covariates such as blood
levels such as fasting glucose and albumin tend to be even better at predicting death than just looking at
methylation alone. Currently, the best biomarkers seem to be C-reactive protein, insulin, fasting glucose,
triglyceride, and high-density lipoprotein (HDL) cholesterol levels.

Methylation Biomarkers + Demographics:

Even more recently, in 2019, a test called GrimAge was published by Dr. Steve Horvath (One of the creators of
the original Biological age clocks). This is probably the best death prediction calculator to date.

Instead of using biomarkers in his calculation, he trained blood biomarkers against the epigenetic data. In short,
he created Biomarkers that could be read from methylation data. This was a vast improvement and allowed for
the best measurement of death prediction.

Beyond lifespan prediction, Age Acceleration with GrimAge (and several of its underlying surrogate biomarkers
including DNAm PAI-1) relate to many age-related conditions (multi-morbidity, metabolic syndrome, markers of
inflammation) in an expected way, i.e. high values are associated with a bad risk profile.

Soon TruAge™ will have a similar test!

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Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

JANE’S TRUAGE™
Treatment Framework

Fitness
- Regularly perform physical (aerobic and
resistance) exercise

- This is more important the older you are

as epigenetic changes are more likely as
you are older
Nutrition
- Epigenetic age changes are correlated
with the level of activity. - Increase fish and poultry
consumption

- Consider a Mediterranean
diet with the help of a
nutritional professional

- Consider a calorie restriction

diet with the help of a
nutritional professional
Medications Drugs/Alcohol
- Consider increasing
- Ask your doctor about - Consider regular consumption consumption of polyphenols
growth hormone optimization of small quantities of wine such as trans-resveratrol,
to regenerate the thymus and beer. sulforaphane, epigallocate
- Consider asking your chin-3-gallate (EGCG),
doctor about DHEA and - Avoid all other recreational quercetin, and genistein.
metformin drugs

- Rapamycin and other

senolytics might be considered
in the future as well as
sirtuin activators Exposures (Toxins, Pollution)
- Stem cell or young plasma
- Wear a mask in highly polluted areas
apheresis procedures
- Spend more time in nature

- Avoid exposure to pesticides and pesticide

treated foods without washing

Comorbidities
Psychosocial
- Avoid behaviors which increase risks of viruses
- Reduce physical and
emotional stress - Avoid type 2 diabetes and obesity

- Seek a counselor for any - Avoid insomnia by creating good sleep habits
PTSD related events

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

CURRENT QUESTION
And Our Investigations Into The Answers!

The study of epigenetics is very new. Thus far only 2 studies have documented how interventions can reverse
epigenetic age. However, we want to change that!
Below we have documented some questions and the studies we are completing to find out the answers!

NAD+
NAD+ is the second most popular cofactor in the human body. NAD+ activate PARPS, Sirtuins, and help with
immune dysregulation and as a result has been viewed as an essential cofactor to help address the process of aging.

The clinical importance of maintaining cellular NAD+ levels was established early in the last century with the
finding that pellagra, a disease characterized by diarrhea, dermatitis, dementia and death, could be cured with
foods containing the NAD+ precursor niacin. Additionally, cellular concentrations of NAD+ have been shown to
decrease under conditions of increased oxidative damage such as occur during aging . Altered levels of NAD+
have been found to accompany several disorders associated with increased oxidative/free radical damage
including diabetes, heart disease, age-related vascular dysfunction, ischemic brain injury, misfolded neuronal
proteins, and Alzheimer’s dementia .

Interventions targeted at restoring NAD+ have been shown in animal models to support healthy aging and
improve metabolic function, and dementia as well.

We have set up a trial as listed below:

Intravenous NAD+ and its effect on Immune Biomarkers and Epigenetic Aging.

In this trial, we will explore how NAD+ effects the bodies cellular immune responses and look how periodic IV
administration can effect epigenetic age.

Senolytics
Cellular senescense is considered to be another of the 8 hallmarks of aging. Senescence is defined as stable
cell growth arrest. Usually this is essential to ensure that damaged or transformed cells do not perpetuate their
genomes. However, when large quantities of cells do this, they can display a Senescence Associated Secretory
Phenotype which can cause a process called inflammaging. This process is highly correlated with most age
related diseases.

Several products have shown to help clear senescent cells from the body and several have been shown to
increase lifespan. One of these products is Rapamycin. We are investigating if this product can help reduce
inflammatory biomarkers and epigenetic age!

We have set up a trial as listed below:

The Safety and Effectiveness of Rapamycin on the Epigenetic Aging Rate in Healthy Individuals

We have seen evidence that Rapamycin slows cell proliferation and decelerates aging and the risk of age -
related diseases. The aim of our pilot study is to evaluate the safety, efficacy and feasibility of Rapamycin as an
effective treatment option to improve clinical care of healthy individual’s biological age thus prolonging longevity.

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

CURRENT QUESTION
And Our Investigations Into The Answers!

Testosterone Replacement and Bariatric Surgery

Andropause is the slow decline in hormone levels in men. Many men try and treat this condition with hormone
replacement therapy. Additionally, as we have detailed it the this report, weight loss is correlated to changes
in epigenetic age. As a result, we will be looking at how both of these things can address epigenetic age both
together and independently through the following study.

#14169, Prospective randomized comparative study to learn if lean muscle catabolism be prevented in
bariatric patients by the use of Hormone Replacement Therapy (HRT) vs patients receiving Standard of Care
(SOC) and how does bariatric surgery with/without HRT impact DNA methaylation-based biomarkers for aging

Young Plasma
Several decades ago, several studies were published showing that when old and young mice were connected
together to share blood, the older mice has several aging benefits and that vice-versa, Young mice became
a little older. A recent revival of this approach confirmed this observation and demonstrated beneficial effects
on muscle, heart, brain, and numerous other organs. Recently, this effect has been investigated in mice by
transferring young mice plasma to older mice and showed that it reduced epigenetic age (less in the brain) in
most systems.

This procedure has also been done by one of our Scientific advisory board members to investigate cognitive
function in humans. We will be conducting some of these procedures again and measurements of sensecence,
immune function, and biological age in the study below!

“Safety and efficacy of Young Plasmapheresis in elderly patients and its effect on cognitive function and
Epignetic biological age”

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

CURRENT QUESTION
And Our Investigations Into The Answers!

Mitochondrial Function
The role of mitochondria in aging was first proposed more than 40 years ago. This theory, the free radical theory
of aging, suggested that accumulation of cellular damage with increasing age results from reactive oxygen
species (ROS) and mitochondria are one of the most important sources and targets of ROS and could function
as an ‘aging clock’.

Since then, a growing body of evidence has shown that mitochondrial dysfunction contributes to aging in
multiple model organisms and that several factors cause increased mitochondrial dysfunction with chronological
age including accumulation of somatic mtDNA mutations, enhanced oxidative damage, decreased abundance
and quality of mitochondria, as well as dysregulation of mitochondrial dynamics.

As mitochondrial dysfunction is one of the hallmarks of aging, we want to look at the effects of mitochondrial
peptides such as SS-31 and MOTS-C have on epigenetic aging.

We will be using these products in patients for 6 months and looking at the effects of mitochondrial functioning
and epigenetic age!

“Mitochondrial peptides and their effect on biomarkers, mitochondrial function, sarcopenia, and biological aging”

Chronic Inflammatory Response Diagnostic (CIRS)

Chronic Inflammatory Response Syndrome (CIRS) is a multi-symptom, multi-system illness caused by exposure to
biotoxins, or neurotoxins produced from a biological source. Often, this test is hard to diagnosis. As a result, we
will be investigating biomarkers correlated to patients diagnosed with this disease in order to develop methylation
markers which might correlate to disease and disease progression. We hope to be able to find highly correlative
markers that could diagnosis this disease at a fraction of the cost of conventional testing.

“Investigation of methylation biomarkers of Chronic inflammatory Response Syndrome versus the normal population”

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

HELP US DESIGN A BETTER TEST

and get your report and analysis for FREE!

One of the best parts about our test is the amount of data we look into! By getting one of the largest
glimpses of your DNA methylation data available, we are able to correlate these to health outcomes and
provide you more insight into your markers! However, in order to create new markers, we need your help!

If you submit any of the following VO2 Max

markers, we will give you a $10
credit toward a future test. Skin Glycosylation Measurements
MRI Imaging
We will also give you a free report
if we use your help in developing Genomic Sequencing
any additional test. Telomere Testing

If you have any of the following Blood and Hormone Levels

testing done within 6 months of
Microbiome Analysis
our test, please upload it to your
patient portal! Proteomic Data

FUTURE TEST

Telomere Estimation Cardiovascular Disease Risk

Cancer Risk
Senescence Burden Estimation

Best Fitness and Diet Estimator

Alzheimer’s Risk

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

KEY TERMS
& Abbreviation

CMV (cytomegalovirus): a common virus that typically only causes problems for immunocompromised and
pregnant individuals

CpG: regions of DNA where a cytosine (C) nucleotide is next to a guanine (C) nucleotide. It is sited in the DNA
where methylation does occur.
CR (Calorie Restrictive): type of diet when you reduce the average daily caloric intake below what is normal
for that particular individual
CRP (C-Reactive Protein): a protein made by the liver, measuring amounts of it in the blood is used to detect
inflammation
DHA (docosahexaenoic acid): an omega-3 fatty acid that’s a structural component of the brain, cerebral
cortex, skin, and retina
DHEA (dehydroepiandrosterone): an endogenous steroid hormone that decreases gradually in adults, that
can act as a precursor to testosterone, estrogen, as a neurosteroid
DNA Methylation: where methyl groups are added to the DNA and can change the expression of that segment
without altering the sequence
EGCG (epigallocatechin-3-gallate): compound used for preventing certain diseases by targeting epigenetic
alterations
EEAA (Extrinsic epigenetic Age Acceleration): aims to measure aging in immune-related components; it also
relates age-associated changes in blood cell composition.
EPA (eicosapentaenoic acid): an omega-3 fatty acid that is prescribed to reduce triglyceride levels
Epigenome: all of the chemical modifications to DNA that regulates the expression of genes
EWAS (epigenome-wide association studies): analyzes epigenetic markers, typically a DNA methylation
marker, to derive epigenetic variations and a particular phenotype
FMD (fasting Mimicking diet): developed by Dr. Valter Longo, it is a 5-day diet that guides the body into a
fasting state, similar to long fasts, while eating quantified meals
GH (growth hormone): a peptide hormone that stimulates growth, development, cell reproduction, and cell
regeneration
HDL (high-density lipoprotein): known as the “good” cholesterol because it removes other forms of cholesterol
from the body
HIV (human immunodeficiency virus): this is a family of viruses that damage to the immune system, which
inhibits the body to fight off infections
Hypogonadism: the testes are no longer to produce testosterone, sperm, or both
Hypothyroidism: a condition where the thyroid gland doesn’t produce enough of the body’s essential hormones
IGF-1 (Insulin-like growth factor-1): a hormone that promotes bone and tissue growth

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 Jane Doe | Test: TruAge™ | Sample ID: BL305 | Collection Date: 2/21/20 | Report Date: 4/12/20

KEY TERMS
& Abbreviation

IEAA (Intrinsic Epigenetic Age Acceleration): a measure of the “pure” epigenetic aging effects in blood cells
that are not confounded by differences in blood cell counts
ORA (Cooperative Health Research in the Region Augsburg): a regional-research platform for population-
based studies in epidemiology and health care research
LMR ( lymphocyte-to-monocyte ratio): reflects systematic inflammation in several tumors
NAS (Normative-aging Study): a longitudinal study on the effects of aging covering a variety of health issues
Metformin: a diabetes medication that regulates blood sugar levels
Nephropathy: a kidney disease caused by damage to the small blood vessels
PAI-1 (Plasminogen activator inhibitor-1): a biomarker for multiple age-related conditions
PM2.5 (PM2.5 PM2.5): refers to atmospheric particulate matter (PM) with a diameter less than 2.5 micrometers
Polyphenols: micronutrients packed with antioxidants and other health benefits
PTSD (post-traumatic stress disorder): mental health disorder that is triggered by a terrifying event that an
individual either experienced or witnessed
Resveratrol: a group of polyphenols; acts like antioxidants by protecting the body against damage that puts
one at risk for diseases like cancer and heart disease
SAAF (Strong African American Families Program): an intervention program meant to improve supporting
parent relationships
Sarcopenia: syndrome marked by the general loss of skeletal muscle mass and strength
Segmental progeria: a rare hereditary disease that’s symptoms resemble enhanced aging; most who are
diagnosed don’t live past their teens
Senescent cells: a state where certain cells are no longer able to divide; this arrest mechanism acts as a
protectant against cancer
SES (Socioeconomic Status): social standing or class of an individual; combines education, income, and
occupation
TFFF (thymic fat-free fraction): measures the functional thymus tissue
Toxoplasmosis: a disease that results from infection with the toxoplasma gondii parasite, which is one of the
world’s most common parasites
Triglyceride: a chemical ester of glycerol and three fatty acids
TRIMM study (Triggers and Mechanisms of Myocardial Infarction): a study of the factors associated with the
transition from chronic coronary artery disease to acute myocardial infarction
UNESCO (United Nations Education, Scientific, and Cultural Organization): this is a specialized agency of the
United Nations

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and coronary heart disease. Genome biology, 17(1), 171. https://doi.org/10.1186/s13059-016-1030-0
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